Cytoplasmic transfer to de-differentiate recipient cells

ABSTRACT

Methods for de-differentiating or altering the life-span of desired “recipient” cells, e.g., human somatic cells, by the introduction of cytoplasm from a more primitive, less differentiated cell type, e.g., oocyte or blastomere are provided. These methods can be used to produce embryonic stem cells and to increase the efficiency of gene therapy by allowing for desired cells to be subjected to multiple genetic modifications without becoming senescent. Such cytoplasm may be fractionated and/or subjected to subtractive hybridization and the active materials (sufficient for de-differentiation) identified and produced by recombinant methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 14/302,384 filed Jun. 11, 2014, now issued as U.S. Pat. No.9,580,683; which is a continuation application of U.S. application Ser.No. 13/617,988 filed Sep. 14, 2012, now abandoned; which is acontinuation application of U.S. application Ser. No. 13/366,518 filedFeb. 6, 2012, now abandoned; which is a continuation application of U.S.application Ser. No. 10/831,599 filed Apr. 23, 2004, now abandoned;which is a continuation application of U.S. application Ser. No.09/736,268 filed Dec. 15, 2000, now abandoned; which is a 35 USC §371National Stage application of International Application No.PCT/US00/18063 filed Jun. 30, 2000, now expired; which claims thebenefit under 35 USC §119(e) to U.S. application Ser. No. 60/141,250filed Jun. 30, 1999, now expired. The disclosure of each of the priorapplications is considered part of and is incorporated by reference inthe disclosure of this application.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods for “de-differentiating” and/oraltering the life-span of desired recipient cells, preferably humansomatic cells. These methods have application especially in the contextof cell therapies and the production of genetically modified cells.

Background Information

Nuclear transfer first gained acceptance in the 1960's with amphibiannuclear transplantation. (Diberardino, M. A. 1980, “Genetic stabilityand modulation of metazoan nuclei transplanted into eggs and ooctyes”,Differentiation, 17-17-30; Diberardino, M. A., N. J. Hoffner and L. D.Etkin, 1984; “Activation of dormant genes in specialized cells”,Science, 224:946-952; Prather, R. S. and Robl, J. M., 1991, “Cloning bynuclear transfer and splitting in laboratory and domestic animalembryos”, In: Animal Applications of Research in Mammalian Development,R. A. Pederson, A. McLaren and N. First (ed.), Cold Spring HarborLaboratory Press.) Nuclear transfer was initially conducted inamphibians in part because of the relatively large size of the amphibianoocyte relative to that of mammals. The results of these experimentsindicated to those skilled in the art that the degree of differentiationof the donor nucleus was greatly instrumental, if not determinative, asto whether a recipient oocyte containing such cell or nucleus couldeffectively reprogram said nucleus and produce a viable embryo.(Diberardino, M. A., N J. Hoffner and L. D. Etkin, 1984, “Activation ofdormant genes in specialized cells.”, Science, 224:946-952; Prather, R.S. and Robl, J. M., 1991, “Cloning by nuclear transfer and splitting inlaboratory and domestic animal embryos”, In: Animal Applications ofResearch in Mammalian Development, R. A. Pederson, A. McLaren and N.First (ed.), Cold Spring Harbor Laboratory Press.)

Much later, in the mid 1980s, after microsurgical techniques had beenperfected, researchers investigated whether nuclear transfer could beextrapolated to mammals. The first procedures for cloning cattle werereported by Robl et al (Robl, J. M., R. Prather, F. Barnes, W. Eyestone,D. Northey, B. Gilligan and N. L. First, 1987, “Nuclear transplantationin bovine embryos”, J. Anim. Sci. 64:642-647). In fact, Dr. Robl's labwas the first to clone a rabbit by nuclear transfer using donor nucleifrom earlier embryonic cells (Stice, S. L. and Robl, J. M., 1988,“Nuclear reprogramming in nuclear transplant rabbit embryos”, Biol.Reprod., 39:657-664). Also, using similar techniques, bovines (Prather,R. S., F L. Barnes, M L. Sims, Robl, J. M., W. H. Eyestone and N. L.First, 1987, “Nuclear transplantation in the bovine embryo: assessmentof donor nuclei and recipient oocyte”, Biol. Reprod., 37:859-866) andsheep (Willadsen, S. M., 1986, “Nuclear transplantation in sheepembryos”, Nature (Lond) 320:63-65), and putatively porcines (Prather, R.S., M. M. Sims and N. L. First, 1989, “Nuclear transplantation in pigembryos”, Biol. Reprod., 41:414), were cloned by the transplantation ofthe cell or nucleus of very early embryos into enucleated oocytes.

In the early 1990s, the possibility of producing nuclear transferembryos with donor nuclei obtained from progressively moredifferentiated cells was investigated. The initial results of theseexperiments suggested that when an embryo progresses to the blastocyststage (the embryonic stage where the first two distinct cell lineagesappear) that the efficiency of nuclear transfer decreases dramatically(Collas, P. and J. M. Robl, 1991, “Relationship between nuclearremodeling and development in nuclear transplant rabbit embryos”, Biol.Reprod., 45:455-465). For example, it was found that trophectodermalcells (the cells that form the placenta) did not support development ofthe nuclear fusion to the blastocyst stage. (Collas, P. and J. M. Robl,1991, “Relationship between nuclear remodeling and development innuclear transplant rabbit embryos”, Biol. Reprod., 45:455-465.) Bycontrast, inner cell mass cells (cells which form both somatic and germline cells) were found to support a low rate of development to theblastocyst stage with some offspring obtained. (Collas P, Barnes F L,“Nuclear transplantation by microinjection of inner cell mass andgranulosa cell nuclei”, Mol Reprod Devel., 1994, 38:264-267.) Moreover,further work suggested that inner cell mass cells which were culturedfor a short period of time could support the development to term. (SimsM, First N L, “Production of calves by transfer of nuclei from culturedinner cell mass cells”, Proc Natl Acad Sci, 1994,91:6143-6147.)

Based on these results, it was the overwhelming opinion of those skilledin the art at that time that observations made with amphibian nucleartransfer experiments would likely be observed in mammals. That is tosay, it was widely regarded by researchers working in the area ofcloning in the early 1990's that once a cell becomes committed to aparticular somatic cell lineage that its nucleus irreversibly loses itsability to become “reprogrammed”, i.e., to support full term developmentwhen used as a nuclear donor for nuclear transfer. While the exactmolecular explanation for the apparent inability of somatic cells to beeffectively reprogrammed was unknown, it was hypothesized to be theresult of changes in DNA methylation, histone acetylation and factorscontrolling transitions in chromatin structure that occur during celldifferentiation. Moreover, it was believed that these cellular changescould not be reversed.

Therefore, it was quite astounding that in 1998, the Roslin Institutereported that cells committed to somatic cell lineage could supportembryo development when used as nuclear transfer donors. Equallyastounding, and more commercially significant, the production oftransgenic cattle which were produced by nuclear transfer usingtransgenic fibroblast donor cells was reported shortly thereafter byscientists working at the University of Massachusetts and Advanced CellTechnology.

Also, recently two calves were reportedly produced at the IshikawaPrefecture Livestock Research Centre in Japan from oviduct cellscollected from a cow at slaughter. (Hadfield, P. and A. Coghlan,“Premature birth repeats the Dolly mixture”, New Scientist, Jul. 11,1998.) Further, Jean-Paul Renard from INTRA in France reported theproduction of a calf using muscle cells from a fetus. (MacKenzie, D. andP. Cohen, 1998, “A French calf answers some of the questions aboutcloning”, New Scientist, March 21.) Also, David Wells from New Zealandreported the production of a calf using fibroblast donor cells obtainedfrom an adult cow. (Wells, D. N., 1998, “Cloning symposium:Reprogramming Cell Fate—Transgenesis and Cloning,” Monash MedicalCenter, Melbourne, Australia, Apr. 15-16.)

Differentiated cells have also reportedly been successfully used asnuclear transfer donors to produce cloned mice. (Wakayama T, Perry A CF, Zucconi M, Johnsoal K R, Yanagimachi R., “Full-term development ofmice from enucleated oocytes injected with cumulus cell nuclei”, Nature,1998, 394:369-374.)

Still further, an experiment by researchers at the University ofMassachusetts and Advanced Cell Technology was recently reported in alead story in the New York Times, January 1999, wherein a nucleartransfer fusion embryo was produced by the insertion of an adultdifferentiated cell (cell obtained from the cheek of an adult humandonor) into an enucleated bovine oocyte. Thus, it would appear, based onthese results, that at least under some conditions differentiated cellscan be reprogrammed or de-differentiated.

Related thereto, it was also recently reported in the popular press thatcytoplasm transferred from oocyte of a young female donor “rejuvenated”an oocyte of an older woman, such that it was competent forreproduction.

However, it would be beneficial if methods could be developed forconverting differentiated cells to embryonic cell types, without theneed for cloning, and the production of embryos, especially given theirpotential for use in nuclear transfer and for producing differentdifferentiated cell types for therapeutic use. Also, it would bebeneficial if the cellular materials responsible for de-differentiationand reprogramming of differentiated cells could be identified andproduced by recombinant methods, thereby improving the efficiency ofcellular reprogramming.

OBJECTS OF THE INVENTION

Therefore, it is an object of the invention to provide novel methods for“de-differentiating” and/or altering the life-span of desired cells.

It is a more specific object of the invention to provide a novel methodfor “de-differentiating” and/or altering the life-span of a desireddifferentiated cell by introducing the cell or cell nucleus withcytoplasm and then transplanting the de-differentiated nucleus into asurrogate cytoplast such as from an ES cell of a less differentiatedcell, preferably an oocyte or blastomere, or another embryonic celltype.

It is another object of the invention to alter the life-span and/or tode-differentiate desired cells, typically mammalian differentiatedcells, prior, concurrent, or subsequent to genetic modification.

It is another object of the invention to provide an improved method ofcell therapy wherein the improvement comprises administering cells whichhave been de-differentiated or have an altered life-span by theintroduction of cytoplasm obtained from a cell of a less orundifferentiated state, preferably an oocyte or blastomere or placingnuclei from said somatic cell into a solution containing an extract ofthe oocyte or blastomere embryo, or ES cell or purified proteins fromthe same.

It is still another object of the invention to identify the component orcomponents in oocyte cytoplasm responsible for de-differentiation and/oralteration of cell life-span, e.g., by fractionation or subtractivehybridization, i.e., fractionation of protein, RNA or DNA.

It is still another object of the invention to provide a novel method oftherapy, especially of the skin, by administering a therapeuticallyeffective amount of cytoplasm obtained from a substantiallyundifferentiated or undifferentiated cell, preferably an oocyte orblastomere, or the purified active components of the same.

It is another object of the invention to provide novel compositions fortherapeutic, dermatologic and/or cosmetic usage that contain cytoplasmderived from substantially undifferentiated or undifferentiated cells,preferably an oocyte or blastomere, or purified active components ofsame.

It is another object of the invention to provide cells for use in celltherapy which have been “de-differentiated” or have an altered life-spanby the introduction of cytoplasm from a substantially undifferentiatedor undifferentiated cell, preferably an oocyte or blastomere, orpurified active components of same.

It is still another object of the invention to provide an improvedmethod of cloning via nuclear transfer wherein the improvement comprisesusing as the donor cell or nucleus a cell which has beende-differentiated and/or has had its life-span altered by theintroduction of cytoplasm from a substantially undifferentiated orundifferentiated cell, or purified active components of same, orcross-species NT where the purified active component is expressed tofacilitate reprogramming.

It is another object of the invention to rejuvenate nuclei isolated fromdesired differentiated cells by contacting same with cytoplasm fromoocytes, blastomeres, ES, or other embryonic cell types.

It is another object of the invention to provide screening assays toidentify proteins, or nucleic acid sequences that are released fromdifferentiated cell nuclei upon contacting with cytoplasm, or fractionsderived from oocyte cytoplasm from oocytes, blastomeres, ES cells orother embryonic cell types, that are involved in all reprogramming.

It is another specific object of the invention to provide screeningassays, e.g., differential or subtractive hybridization to identifymRNAs that expressed in oocyte cytoplasm or in embryonic cell types thatare involved in cell programming.

SUMMARY OF THE INVENTION

The present invention provides novel methods for producing cells,preferably mammalian cells and, most preferably, human cells that havebeen de-differentiated and/or which have an altered (increased)life-span by the juxtaposition of the donor cell with cytoplasm from anundifferentiated or substantially undifferentiated cell, preferably anoocyte or blastomere, or another embryonic cell type. In a particularlypreferred embodiment, the present invention will be used to producecells in a more primitive state, especially embryonic stem cells orinner cell mass cells.

The resultant cells are useful in gene and cell therapies, and as donorcells or nuclei for use in nuclear transfer.

DEFINITIONS

“Ooctye”—In the present invention, this refers to any oocyte, preferablya mammalian oocyte, that develops from an oogonium and, followingmeiosis, becomes a mature ovum.

“Metaphase II Ooctye”—The preferred stage of maturation of oocytes usedfor nuclear transfer (First and Prather, Differentiation, 48:1-8). Atthis stage, the oocyte is sufficiently “prepared” to treat an introduceddonor cell or nucleus as it does a fertilizing sperm.

“Donor Cell”—In the present invention, this refers to a cell whereinsome or all of its cytoplasm is transferred to another cell (“recipientcell”). The donor cell is typically a primitive or embryonic cell type,preferably an oocyte, blastomere, or inner cell mass cell.

“Recipient Cell”—This refers to a cell into which all or part of thecytoplasm of a donor cell, wherein such donor cell is of a moreprimitive cell type relative to the recipient cell, is transferred. Thistransfer can be accomplished by different methods, e.g., microinjectionor by contacting donor cells with liposomal encapsulated cytoplasm orenucleating the donor cell and incubating with cytoplasmic extract.Typically, the donor cell is an oocyte, blastomere or inner cell masscell, and the recipient cell is a somatic cell, preferably a humansomatic cell.

“Blastomere”—Embryonic, substantially undifferentiated cells containedin blastocyst stage embryos.

“Embryonic Cell” or “Embryonic Cell Type”—In the present invention, thiswill refer to any cell, e.g., oocyte, blastomere, embryonic stem cell,inner cell mass cell, or primordial germ cell, wherein the introductionof cytoplasm therefrom into a differentiated cell, e.g., human somaticcell in tissue culture, results in de-differentiation and/or lengtheningof the life-span of such differentiated cell.

“Cell Having Altered Life-Span”—In the present invention this refers tothe change in cell life-span (lengthening) that results when cytoplasmof a more primitive or less differentiated cell type, e.g., an embryoniccell or embryonic cell type, e.g., oocyte or blastomere, is introducedinto a desired differentiated cell, e.g., a cultured human somatic cell.

“Embryonic Stem Cell (ES Cell)”—In the present invention this refers toan undifferentiated cell that has the potential to develop into anentire organism, i.e., a cell that is able to propagate indefinitely,maintaining its undifferentiated state and, when induced todifferentiate, be capable of giving rise to any cell type of the body.

“Nuclear Transfer”—Introduction of cell or nuclear DNA of donor cellinto enucleated oocyte which cell or nucleus and oocyte are then fusedto produce a nuclear transfer fusion or nucleus fusion embryo. This NTfusion may be used to produce a cloned embryo or offspring or to produceES cells.

“Telomerase”—A ribonucleoprotein (RNP) particle and polymerase that usesa portion of its internal RNA moiety as a template for telomere repeatDNA synthesis (U.S. Pat. No. 5,583,016; Yu et al, Nature, 344:126(1990); Singer and Gottschling, Science, 266:404 (1994); Autexier andGreider, Genes Develop., 8:563 (1994); Gilley et al, Genes Develop.,9:2214 (1995); McEachern and Blackburn, Nature, 367:403 (1995);Blackburn, Ann. Rev. Biochem., 61:113 (1992); Greider, Ann. Rev.Biochem., 65:337 (1996).) The activity of this enzyme depends upon bothits RNA and protein components to circumvent the problems presented byend replication by using RNA (i.e., as opposed to DNA) to template thesynthesis of telomeric DNA. Telomerases extend the G strand of telomericDNA. A combination of factors, including telomerase processivity,frequency of action at individual telomeres, and the rate of degradationof telomeric DNA, contribute to the size of the telomeres (i.e., whetherthey are lengthened, shortened, or maintained at a certain size). Invitro telomerases may be extremely processive, with the Tetrahymenatelomerase adding an average of approximately 500 bases to the G strandprimer before dissociation of the enzyme (Greider, Mol. Cell. Biol.,114572 (1991).)

“Genetically Modified or Altered”—In the present invention this refersto cells that contain one or more modifications in their genomic DNA,e.g., additions, substitutions and/or deletions.

“De-Differentiation”—In the present invention, this refers to thechanges in a differentiated cell, e.g., human somatic cell in tissueculture, that result upon introduction of cytoplasm from a moreprimitive, less differentiated cell type, e.g., an oocyte or otherembryonic cell.

“Totipotent”—In the present invention this refers to a cell that givesrise to all of the cells in a developing body, such as an embryo, fetus,an animal. The term “totipotent” can also refer to a cell that givesrise to all of the cells in an animal. A totipotent cell can give riseto all of the cells of a developing cell mass when it is utilized in aprocedure for creating an embryo from one or more nuclear transfersteps. An animal may be an animal that functions ex utero. An animal canexist, for example, as a live born animal. Totipotent cells may also beused to generate incomplete animals such as those useful for organharvesting, e.g., having genetic modifications to eliminate growth of ahead such as by manipulation of a homeotic gene.

“Ungulate”—In the present invention this refers to a four-legged animalhaving hooves. In other preferred embodiments, the ungulate is selectedfrom the group consisting of domestic or wild representatives of bovids,ovids, cervids, suids, equids, and camelids. Examples of suchrepresentatives are cows or bulls, bison, buffalo, sheep, big-hornsheep, horses, ponies, donkeys, mule, deer, elk, caribou, goat, waterbuffalo, camels, llama, alpaca, and pigs. Especially preferred in thebovine species are Bos taurus, Bos indicus, and Bos buffaloes cows orbulls.

“Immortalized” or “Permanent”Cell—These terms as used in the presentinvention in reference to cells can refer to cells that have exceededthe Hayflick limit. The Hayflick limit can be defined as the number ofcell divisions that occur before a cell line becomes senescent, Hayflickset this limit to approximately 60 divisions for most non-immortalizedcells (See, e.g., Hayflick and Moorhead, 1971, Exp. Cell. Res.,25:585-621; and Hayflick, 1965, Exp. Cell Research, 37:614-636,incorporated herein by reference in their entireties, including allfigures, tables and drawings.) Therefore, an immortalized cell line canbe distinguished from non-immortalized cell lines if the cells in thecell line are able to undergo more than 60 divisions. If the cells of acell line are able to undergo more than 60 cell divisions, the cell lineis an immortalized or permanent cell line. The immortalized cells of theinvention are preferably able to undergo more than 70 divisions, aremore preferably able to undergo more than 90 divisions, and are mostpreferably able to undergo more than 90 cell divisions.

Typically, immortalized or permanent cells can be distinguished fromnon-immortalized and non-permanent cells on the basis that immortalizedand permanent cells can be passaged at densities lower than those ofnon-immortalized cells. Specifically, immortalized cells can be grown toconfluence (e.g., when a cell monolayer spreads across an entire plate)when plating conditions do not allow physical contact between the cells.Hence, immortalized cells can be distinguished from non-immortalizedcells when cells are plated at cell densities where the cells do notphysically contact one another.

“Culture”—In the present invention this term refers to one or more cellsthat are static or undergoing cell division in a liquid medium. Nearlyany type of cell can be placed in cell culture conditions. Cells may becultured in suspension and/or in monolayers with one or moresubstantially similar cells. Cells may be cultured in suspension and/orin monolayers with heterogeneous population cells. The termheterogeneous as utilized in the previous sentence can relate to anycell characteristics, such as cell type and cell cycle stage, forexample. Cells may be cultured in suspension and/or in monolayers withfeeder cells.

“Feeder Cells”—This refers to cells grown in co-culture with othercells. Feeder cells include, e.g., fibroblasts, fetal cells, oviductalcells, and may provide a source of peptides, polypeptides, electricalsignals, organic molecules (e.g., steroids), nucleic acid molecules,growth factors, cytokines, and metabolic nutrients to cells co-culturedtherewith. Some cells require feeder cells to be grown in tissueculture.

“Reprogram”—This term as used in the present invention refers tomaterials and methods that can convert a differentiated cell into a lessdifferentiated, more primitive cell type, e.g., an embryonic stem cell.

“Embryo”—In the present invention this refers to a developing cell massthat has not implanted into the uterine membrane of a maternal host.Hence, the term “embryo” as used herein can refer to a fertilizedoocyte, a cybrid (defined herein), a pre-blastocyst stage developingcell mass, and/or any other developing cell mass that is at a stage ofdevelopment prior to implantation into the uterine membrane of amaternal host. Embryos of the invention may not display a genital ridge.Hence, an “embryonic cell” is isolated from and/or has arisen from anembryo.

“Fetus”—In the present invention refers to a developing cell mass thathas implanted into the uterine membrane of a maternal host. A fetus caninclude such defining features as a genital ridge, for example. Agenital ridge is a feature easily identified by a person of ordinaryskill in the art and is a recognizable feature in fetuses of most animalspecies.

“Fetal Cell”—as used herein can refer to any cell isolated from and/orhas arisen from a fetus or derived from a fetus,

“Non-Fetal Cell”—refers to a cell that is not derived or isolated from afetus.

“Senescence”—In the present invention this refers to the characteristicslowing of growth of non-immortal somatic cells in tissue culture aftercells have been maintained in culture for a prolonged period.Non-immortal cells characteristically have a defined life-span beforethey become senescent and die. The present invention alleviates orprevents senescence by the introduction of cytoplasm from a donor cell,typically an oocyte or blastomere, into a recipient cell, e.g., acultured human somatic cell.

DETAILED DESCRIPTION OF THE INVENTION

As explained above, the present invention provides novel methods forde-differentiating and/or altering the life-span of desired cells,preferably mammalian cells and, most preferably, human or other primatecells by the introduction of cytoplasm from a more primitive cell type,typically an undifferentiated or substantially undifferentiated cell,e.g., an oocyte or blastomere.

As noted previously, it was recently reported in the popular press thata group working in the area of artificial insemination and infertilitysuccessfully transferred the cytoplasm from the oocyte of a youngerwoman into that of an older woman and thereby rejuvenated the ability ofthe older oocyte to be competent for fertilization and embryodevelopment. Based on this anecdotal evidence, coupled with recentpapers in the scientific literature which suggest that differentiatedadult cells may be effectively “reprogrammed” by nuclear transfer, itwas theorized that differentiated cells could be effectively“reprogrammed” or “de-differentiated” and/or have their life-spanaltered (increased) by the introduction of cytoplasm from that ofundifferentiated or substantially undifferentiated cell, e.g., an oocyteor blastomere or another embryonic cell type.

While it is presently unknown how the cytoplasm of one cell affects thelife-span or state of differentiation of another, it is theorized thatthe cytoplasm of cells in early or primitive states of developmentcontains one or more substances, e.g., transcription factors and/orother substances that act to trigger or promote cell differentiation.For example, one substance likely contained therein that affects thestate of cell differentiation is telomerase. Another substance is OCT-4and REX. However, Applicant does not wish to be bound to this theory asit is not necessary for an understanding of the invention.

In the present invention, a recipient cell will typically bededifferentiated in vitro by the introduction of an effective amount ofcytoplasm from a donor cell, i.e., an undifferentiated or substantiallyundifferentiated cell, e.g., an oocyte or blastomere. This introductionor transfer of cytoplasm can be effected by different methods, e.g., bymicroinjection or by use of a liposomal delivery system. A preferredmeans comprises the introduction of cytoplasm blebs derived from EScells, oocytes or other embryonic cells into desired differentiatedcells, e.g., mammalian or other cells which are at or near senescence.For example, such cytoplasm blebs can be introduced into geneticallymodified mammalian cells in order to rejuvenate such cells, e.g., priorto their usage for cell therapy.

Alternatively, cytoplasmic blebs can be contacted with nuclei fromdifferentiated cells to induce rejuvenation.

The recipient cell can be of any species and may be heterologous to thedonor cell, e.g., amphibian, mammalian, avian, with mammalian cellsbeing preferred. Especially preferred recipient cells include human andother primate cells, e.g., chimpanzee, cynomolgus monkey, baboon, otherOld World monkey cells, caprine, equine, porcine, ovine, and otherungulates, murine, canine, feline, and other mammalian species.

Also, the recipient cell can be any differentiated cell type. Suitableexamples thereof include epithelial cells, endothelial cells,fibroblasts, keratinocytes, melanocytes and other skin cell types,muscle cells, bone cells, immune cells such as T and B-lymphocytes,oligodendrocytes, dendritic cells, erythrocytes and other blood cells;pancreatic cells, neural and nerve cell types, stomach, intestinal,esophageal, lung, liver, spleen, kidney, bladder, cardiac, thymus,corneal, and other ocular cell types, etc. In general, the methods haveapplication in any application wherein a source of cells that are in aless differentiated state would be desirable.

As noted, the transferred cytoplasm will be obtained from a “donor” cellthat is in a less differentiated state or more primitive state than therecipient cell. Typically, the cytoplasm will be derived from oocytes orcells of early stage embryos, e.g., blastomeres or inner cell mass cellsderived from early stage embryos, in general, it is preferred that thedonor cytoplasm be obtained from oocytes or other embryonic cells thatare in an undifferentiated or substantially undifferentiated state.Bovine oocytes are a preferred source because they can be readilyobtained in large quantities from slaughterhouses.

Recently there have been reports in the literature concerning theproduction of cultures comprising embryonic stem cells that reportedlyexpress or do not express certain markers characteristic of embryonicstem cells. It is therefore also preferable that donor cytoplasm beobtained from an oocyte or other cell that expresses or does not expresscell markers which are characteristic of an undifferentiated, embryoniccell type. Such markers on primate ES cells include, by way of example,SSEA-1 (−); SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); andalkaline phosphatase (+). (See U.S. Pat. No. 5,843,780 to Thomson,issued Dec. 1, 1998.)

As discussed above, it is also desirable that telomerase and/or a DNAsequence or other compound that provides for the expression oftelomerase be introduced into the recipient cell, e.g., a mammalian celland, more preferably, a human or non-human primate cell. The isolationof telomerase and cloning of the corresponding DNA has been reportedprior to the present invention. For example, WO 98/14593, published Apr.9, 1998, by Cech et al, reports telomerase nucleic acid sequencesderived from Eeuplotes aediculatus, Saccharomyces, Schizosaccharomyces,and human, as well as polypeptides comprising telomerase proteinsubunits. Also, WO 98/14592, to Cech et al, published Apr. 9, 1998,discloses compositions containing human telomerase reversetranscriptase, the catalytic protein subunit of human telomerase. Also,U.S. Pat. Nos. 5,837,857 and 5,583,414 describe nucleic acids encodingmammalian telomerases.

Still further, U.S. Pat. No. 5,830,644, issued to West et al; U.S. Pat.No. 5,834,193, issued to Kzolowski et al, and U.S. Pat. No. 5,837,453,issued to Harley et al, describe assays for measuring telomerase lengthand telomerase activity and agents that affect telomerase activity.These patents and PCT applications are incorporated by reference intheir entirety herein.

Thus, in the present invention, desired cells, e.g., cultured humansomatic cells, may be de-differentiated or reprogrammed in tissueculture by the introduction of cytoplasm of a more primitive cell type,e.g., an oocyte or embryonic cell type alone or in conjunction withtelomerase. The introduction of cytoplasm from a donor oocyte orembryonic cell, e.g., blastomere, may be accomplished by variousmethods. For example, this can be effected by microsurgically removingpart or all of the cytoplasm of a donor oocyte or blastomere or otherembryonic cell type with a micropipette and microinjecting suchcytoplasm into that of a recipient mammalian cell. It may also bedesirable to remove cytoplasm from the recipient cell prior to suchintroduction. Such removal may be accomplished by well-knownmicrosurgical methods. Alternatively, the cytoplasm and/or telomerase ortelomerase DNA can be introduced using a liposomal delivery system.

The present methods should provide a means of producing embryonic stemcells, e.g., mammalian embryonic stem cells, and most desirably, humanembryonic stem cells, by reprogramming or de-differentiating desiredcells in tissue culture. These cells are desirable from a therapeuticstandpoint since such cells can be used to give rise to anydifferentiated cell type. The resultant differentiated cell types may beused in cell transplantation therapies.

Another significant application of the present invention is for genetherapy. To date, many different genes of significant therapeuticimportance have been identified and cloned. Moreover, methods for stablyintroducing such DNAs into desired cells, e.g., mammalian cells and,more preferably, human somatic cell types, are well known. Also, methodsfor effecting site-specific insertion of desired DNAs via homologousrecombination are well known in the art.

However, while suitable vectors and methods for introduction anddetection of specific DNAs into desired somatic cells are known, asignificant obstacle to the efficacy of such methods is the limitedlife-span of normal, i.e., non-immortal cells, in tissue culture. Thisis particularly problematic in situations wherein the introduction ofmultiple DNA modifications, e.g., deletions, substitutions, and/oradditions is desired. Essentially, while methods for effecting targetedDNA modifications are known, the requisite time to effect and select forsuch modifications can be very lengthy. Thus, the cells may becomesenescent or die before the desired DNA modifications have beeneffected.

The present invention will alleviate this inherent constraint of geneand cell therapy by introducing the cytoplasm of an oocyte or otherembryonic cell type into recipient cells prior, concurrent or subsequentto genetic modification. The introduction of such cytoplasm alone or incombination with telomerase or a DNA or another compound that results inthe expression of telomerase, will reprogram the genetically modifiedcell and enable it to have a longer life-span in tissue culture. Suchreprogramming can be effected once or repeatedly during geneticmodification of recipient cells. For example, in the case of verycomplex genetic modifications, it may be necessary to “reprogram”recipient cells several times by the repeated introduction of donorcytoplasm to prevent senescence. The optimal frequency of suchreprogramming will be determined by monitoring the doubling time of thecells in tissue culture such that the cells are reprogrammed before theybecome senescent.

The resultant reprogrammed genetically modified cells, which have alonger life-span as a result of reprogramming, may be used for cell andgene therapy. Moreover, these cells may be used as donor cells fornuclear transfer procedures or for the production of chimeric animals.The present methods will make it possible to produce cloned and chimericanimals having complex genetic modifications. This will be especiallyadvantageous for the production of animal models for human diseases.Also, the present methods will be beneficial in situations wherein theexpression of a desired gene product or phenotype is dependent upon theexpression of different DNA sequences, or for gene research involvingthe interrelated effects of different genes on one another. Moreover, itis anticipated that the present methods will become very important asthe interrelated effects of the expression of different genes on othersbecomes more understood.

Yet another application of the present invention is for alleviating theeffects of aging. Just as mammalian cells have a finite life-span intissue culture, they similarly have a finite life-span in vivo. Thisfinite life-span is hypothesized to explain why organisms, includinghumans, have a normal maximum life-span, determined by the finitelife-span of human somatic cells.

The present invention will alleviate the effects of aging by takingmammalian cells from an individual and altering (lengthening) thelife-span of such cells by introduction of cytoplasm from an oocyte orother embryonic cell type, e.g., blastomere. The resultant rejuvenatedcells may be used to produce differentiated cell types in tissue cultureand these cells can then be introduced into the individual. This can beused, e.g., to rejuvenate the immune system of an individual. Suchrejuvenation should be useful in the treatment of diseases thought to beof immune origin, e.g., some cancers.

Also, the subject methods may be used for the production of autologousgrafts, e.g., skin grafts, which can be used in the case of tissueinjury or elective surgery.

Yet another application of the present application is for treating theeffects of chronologic and UV-induced aging on the skin. As skin ages,various physical changes may be manifested including discoloration, lossof elasticity, loss of radiance, and the appearance of fine lines andwrinkles. It is anticipated that such effects of aging may be alleviatedor even reversed by topical application of cytoplasm-containingcompositions. For example, cytoplasm from donor oocytes, e.g., bovineoocytes, optionally further including telomerase or a telomerase DNAconstruct, can be packaged in liposomes to facilitate internalizationinto skin cells upon topical application. Also, it may be advantageousto include in such compositions compounds that facilitate absorptioninto the skin, e.g., DMSO. These compositions may be topically appliedto areas of the skin wherein the effects of aging are most pronounced,e.g., the skin around the eyes, the neck and the hands.

Still another application of the present invention is for identificationof the substance or substances found in cytoplasm that inducesde-differentiation. This can be effected by fractionation of cytoplasmand screening these fractions to identify those which contain substancesthat result in effective rejuvenation or reprogramming when transferredinto recipient cells, e.g., human differentiated cell types.

Alternatively, the components) contained in oocyte cytoplasm responsiblefor reprogramming or rejuvenation can be identified by sub tractivehybridization by comparing mRNA expression in early stage embryos andoocytes to that of more differentiated embryos.

With respect to such identification, it is currently unknown whatcomponent or compounds contained in embryonic cell cytoplasm areresponsible for cell reprogramming or de-differentiation. In fact, it isuncertain even as to the specific nature of such component(s), e.g.,whether they are nucleic acids or proteinases.

However, it is speculated by the present inventors that suchcomponent(s) may comprise nucleic acids, in particular maternal RNAs, orproteins encoded thereby. In this regard, it has been reported bydifferent groups that very early stage embryos contain a class of RNAknown as maternal RNA's that are stored in the egg very early on butwhich are not detected past the blastula stage.(Kontrogianni-Konstantopoulos et al, Devel. Biol., 177(2):371-382(1996).) Maternal RNA levels have been quantified for different species,i.e., rabbit, cow, pig, sheep and mouse. (Olszanska et al, J. Exp.Zool., 265(3):317-320 (1993).) With respect thereto, it has also beenreported that maternal RNA in Drosophila oocyte encodes a protein thatmay bind to a tyrosine kinase receptor present in adjacent folliclecells that may initiate various events leading to dorsal follicle celldifferentiation which act to delimit and orient the future dorsoventralaxis of the embryo. (Schupbach et al, Curr. Opin. Genet. Dev.,4(4):502-507 (1994).)

Also, fractionation of oocytes has shown that mitogen-activated proteinkinases are expressed at higher levels in small oocytes, suggesting thatit is a maternal RNA that is stored for early embryogenesis. This isspeculated to be involved in signal transduction in embryonic as well asadult cells. (Zaitsevskaya et al, Cell Growth Differ., 3(11):773-782(1992).)

Still further, it has been reported that a maternal mRNA in silkwormoocytes encodes a protein that may be a structural component necessaryfor formation of the cellular blastoderm of the embryo, and that theassociation of such maternal mRNA with cortical cytoskeleton mayparticipate in the synthesis of new cytoskeleton or related structuresduring blastoderm development. (Kastern et al, Devel.,108(3):497-505(1990).)

Moreover, it has been reported that maternal poly(A)+ RNA moleculesfound in the egg of the sea urchin and amphibian oocyte are completedwith U1 RNA, a co-factor in somatic nuclear pre-mRNA splicing and thatsuch RNAs contain repeated sequences interspersed with single-copyelements. (Calzone et al, Genes Devel., 2(3):305-318 (1988); Ruzdijic etal, Development, 101(1):107-116 (1987).)

Thus, based thereon, and the observation that cytoplasm apparentlycontains some component that results in cell reprogramming, it should bepossible to identify compounds, likely nucleic acids and/orproteinaceous compounds which are present in the cytoplasm of oocytesand early embryos that, under appropriate conditions, provide forreprogramming or de-differentiation of desired cells. This will beeffected by fractionation of cytoplasm into different fractions, e.g.,based on size or isoelectric point, and ascertaining those factors whicheffect de-differentiation or reprogramming when transferred todifferentiated cell types.

Alternatively, the factors responsible for reprogramming may beidentified by sub tractive or differential hybridization, essentially byidentifying those mRNAs which are present in oocytes that are lost afterthe embryo has differentiated beyond a certain stage, e.g., past theblastula stage of development, and identifying those of which areinvolved in de-differentiation or reprogramming.

Therefore, the invention includes the identification of the specificcytoplasmic materials, e.g., polypeptides and/or nucleic acid sequences,which when transferred into a differentiated cell provide forde-differentiation or reprogramming. Based on what has been reportedwith respect to maternal RNAs, it is anticipated that the activematerials responsible for de-differentiation or reprogramming mayinclude maternal RNAs or polypeptides encoded thereby.

After such nucleic acid(s) or polypeptides have been identified andsequenced, they will be produced by recombinant methods. It isanticipated that these recombinantly produced nucleic acids orpolypeptides will be sufficient to induce reprogramming orde-differentiation of desired cells.

The invention further encompasses assays wherein oocyte cytomplasm orcytoplasm from ES cells is fractionated into different fractions, e.g.,based on molecular weight, isoelectric point, gel filtration, and saltprecipitation, which are added into different microwells that containone or more isolated nuclei from desired differentiated cells, e.g.,mammalian, amphibian, avian, or insect cells and a screening assayconducted to identify mRNAs such as REX or OCT-4 that are released fromthe nuclei. For example, such mRNAs may be identified by PCRamplification and detection.

Alternatively, PCR screening assays may be conducted' wherein ooplasmcan be added to desired differentiated cells and assays conducted toidentify what mRNAs, e.g., REX or OCT-4, are released from the cellnuclei after introduction of the oocyte cytoplasm.

The identification of such mRNAs can be identified by known methods,e.g., subtractive hybridization, differential display, and differentialhyridization techniques. Essentially, these methods provide for thecomparison of different populations of mRNAs in different cells, orcells at different times, and are conventionally used to identify genesthat are expressed only under specific conditions or by specific typesof cells.

In particular, subtractive hybridization can be effected by use ofoocyte RNAs which are subtracted with RNAs obtained from normal somaticcell RNAs. Thereby, RNAs that are involved in cell reprogramming can beidentified.

Additionally, the invention further includes the reconstitution ofnuclei isolated from desired differentiated cells, e.g., those which arederived from differentiated cells in tissue culture, which potentiallymay be genetically modified by contacting such isolated nuclei withcytoplasm fractionated from oocytes, blastomeres or ES cells, and theaddition of such reconstituted nuclei to cytoplasts, thereby producing arejuvenated cell having increased proliferation potential and lifespan.

What is claimed is:
 1. A method for producing a totipotent cellcomprising: a) transferring all or part of the cytoplasm of a donor cellinto an isolated recipient cell; and b) transferring a telomerase or aDNA construct that provides for the expression of telomerase into therecipient cell or recipient cell nucleus; wherein the donor cell is ator near senescence.
 2. The method of claim 1, wherein the donor cell isless differentiated than the recipient cell or is an undifferentiatedcell.
 3. The method of claim 1, wherein said donor cell is an oocyte oran embryonic cell.
 4. The method of claim 1, wherein the telomerase DNAunder the control of a regulatable promoter.
 5. The method of claim 1,wherein said recipient cell is a mammalian cell.
 6. The method of claim5, wherein said mammalian cell is derived from a mammal selected fromthe group consisting of non-human primate, human, rat, guinea pig,mouse, rabbit, dog, cat, hamster, goat, cattle, sheep, horse, bison andbuffalo.
 7. The method of claim 6, wherein said mammalian cell isselected from the group consisting of cardiac, lung, skin, liver,stomach, intestine, neural, muscle, bone, cartilage, immune, pancreatic,spleen, esophageal, and corneal cells.
 8. The method of claim 1, whereinsaid recipient cell or recipient cell nucleus is genetically modifiedprior, concurrent or subsequent to the introduction of said cytoplasm.9. The method of claim 10, wherein (a) said genetically modified cellscomprise several genetic modifications; or (b) said genetically modifiedrecipient cell or recipient cell nucleus comprises a recombinant DNAthat encodes a desired polypeptide.
 10. The method of claim 1, whichresults in the increased life-span of a mammalian recipient cell orrecipient cell nucleus.
 11. The method of claim 1, wherein said donorcell is of a different species than the recipient cell.
 12. The methodof claim 11, wherein said donor cell is a non-human primate oocyte orembryonic cell and the recipient cell is a human somatic cell.
 13. An invitro method for producing an embryonic stem cell, the methodcomprising: a) providing a nucleus isolated from a cell; b) transferringpart of the cytoplasm of a cytoplasm donor cell into said nucleus; c)introducing a telomerase or a DNA construct that provides for theexpression of telomerase into said nucleus or the cell from which saidnucleus is isolated; and d) introducing said nucleus into a cytoplast.14. The method of claim 13, wherein the embryonic stem cell has anincreased life-span relative to the cell from which said nucleus isisolated, and wherein the donor cell is at or near senescence.
 15. Themethod of claim 13, wherein the cytoplasm is derived from an oocyte orembryonic cell.
 16. The method of claim 13, wherein the telomerase DNAunder the control of a regulatable promoter.
 17. The method of claim 13,wherein the cell from which said nucleus is isolated is a mammaliancell.
 18. The method of claim 13, wherein said recipient cell orrecipient cell nucleus is genetically modified prior, concurrent orsubsequent to the introduction of said cytoplasm.
 19. The method ofclaim 13, wherein said donor cell is of a different species than therecipient cell.
 20. The method of claim 19, wherein said donor cell is anon-human primate oocyte or embryonic cell and the recipient cell is ahuman somatic cell.